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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11/08/2016-26/09/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
9β,11β-epoxy-17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione
EC Number:
213-563-4
EC Name:
9β,11β-epoxy-17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione
Cas Number:
981-34-0
Molecular formula:
C22H28O5
IUPAC Name:
17,21-dihydroxy-16-methyl-9,11-epoxypregna-1,4-diene-3,20-dione
Test material form:
solid: particulate/powder

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The assay on test item was carried out in parallel with the following bottles:
- a known ready biodegradable reference item (one replicate), Sodium Benzoate, at
100 mg/L;
- two inoculum control replicates (blank);
- two test item replicates at 40 mg/L concentration;
- one abiotic control containing the test item at 40 mg/L and a sterilizing agent (HgCl2)
at 100 mg/L (without inoculum);
- one toxicity control, containing the test item at 40 mg/L and reference item at 100
mg/L.
BOD flasks of 500 mL capacity, equipped with pressure sensors fitted on the bottle mouth,
were used as test units.
The method is based on an electro-chemical analyses process. The biodegradation process
consumes the dissolved oxygen in liquid and generates CO2. Evolved carbon dioxide is
adsorbed by potassium hydroxide and the total pressure decreases in the test flasks. The
pressure decrease is detected and converted into an electrical signal by means of an
electrode type manometer.

The test medium was prepared according to the testing guidelines: salts of analytical grade
were dissolved in purified water to obtain the following stock solutions (up to 1000 mL):
a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.42
b) CaCl2 x 2H2O 36.40 g/L
or
CaCl2 27.50 g/L
c) MgSO4 x 7H2O 22.50 g/L
d) FeCl3 x 6 H2O 0.25 g/L
In order to allow the storage of solution, one drop of concentrated HCl was added.
10 mL of stock solution a) and 1 mL of stock solutions b), c) and d) were added to 800 mL of
purified water, then made up to 1000 mL with purified water. The pH of test medium was
7.48 (mean value between two blank bottles), so adjustment with a diluted hydrochloric acid
or sodium hydroxide solution, before making up to volume, was not needed.

Since the test item is not totally soluble in water, the test item was directly weighed into the
test flasks to obtain the final concentrations of 40 mg/L of test item. The test flasks were
made up to a final volume of 250 mL or 150 mL (based on setting scale) with test medium
(see Table 1).
Moreover, stock solutions (10 g/L) of reference item Sodium Benzoate and sterilizing agent
HgCl2 were prepared and an appropriate aliquot of the stock solution was added to the test
medium (final volume: 250 mL or 150 mL ) in the test flasks to obtain a final concentration of
100 mg/L.
Activated sludge was added to each flask (with the exception of the abiotic control) before
incubation.
Finally, the test flasks, containing stir bars covered with teflon, were kept under continuous
stirring by the means of a magnetic stirrer for the 28-days test period.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
40 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The assay on test item was carried out in parallel with the following bottles:
- a known ready biodegradable reference item (one replicate), Sodium Benzoate, at
100 mg/L;
- two inoculum control replicates (blank);
- two test item replicates at 40 mg/L concentration;
- one abiotic control containing the test item at 40 mg/L and a sterilizing agent (HgCl2)
at 100 mg/L (without inoculum);
- one toxicity control, containing the test item at 40 mg/L and reference item at 100
mg/L.
BOD flasks of 500 mL capacity, equipped with pressure sensors fitted on the bottle mouth,
were used as test units.
The method is based on an electro-chemical analyses process. The biodegradation process
consumes the dissolved oxygen in liquid and generates CO2. Evolved carbon dioxide is
adsorbed by potassium hydroxide and the total pressure decreases in the test flasks. The
pressure decrease is detected and converted into an electrical signal by means of an
electrode type manometer.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
A study to evaluate the ready biodegradability of the test item BETA-METHYLEPOXY,
following the “Manometric Respirometry” method, was performed.
The tested concentration was 40 mg/L (corresponding to 91.2 mg ThOD/L).
All reported results are related to the nominal concentration of test item, since the test
concentrations were not measured during the test.
The biodegradation was followed by measuring the oxygen uptake (mg/L) of the test item
under defined conditions by the means of a manometric method and calculating from these
values the BOD (Biochemical Oxygen Demand).
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
0.5
Sampling time:
28 d
Details on results:
The ThODNH3 value (Theoretical Oxygen Demand without nitrification process) was calculated to be equal to 1.67 mg O2/mg for reference item, Sodium Benzoate and equal to 2.28 mg O2/mg for test item.
These values, 1.67 mg O2/mg for reference item and 2.28 mg O2/mg for the test item, were used to calculate the % biodegradation.
Nitrate and nitrite concentration was not measured since the test item does not contain nitrogen.
The test item was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, since no biodegradation was observed during the test period (0.5 % of biodegradation, as mean value between two test item replicates.
No significant degradation was observed in the abiotic control.
The toxicity control clearly showed that the test item does not cause inhibitory effect on the microorganisms activity at the tested concentration. Actually, the toxicity control exceeded
the 25 % pass level after five days of exposure (26.6 %).
The reference item exceeded the biodegradation pass level (60%) after six days (63.2 %), within the range fixed by OECD guideline (first 14 days).

BOD5 / COD results

BOD5 / COD
Parameter:
BOD5
Value:
0.96 mg O2/g test mat.
Results with reference substance:
The ThODNH3 value (Theoretical Oxygen Demand without nitrification process) was
calculated to be equal to 1.67 mg O2/mg for reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item, BETA-METHYLEPOXY was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, since no biodegradation was reached during the test period (0.5% of biodegradation as mean value between two test item replicates). No significant degradation was observed in the abiotic control. Moreover, the test item showed no inhibitory effects on the microorganism activity at the tested concentration, as demonstrated by the biodegradation percentage in the toxicity control exceeding the 25 % pass level after five days of exposure (26.6 %).